Review
Hiroshi Kasai * , Kazuaki Kawai and Yun-Shan Li
Free radical-mediated cytosine C-5 methylation triggers epigenetic changes during
carcinogenesis
Abstract: The methylation of the C-5 position of deoxy- cytidine (dC) in the promoter regions of tumor suppres- sor genes is often observed in cancer cells. We found that various environmental agents, as well as endogenous compounds such as methionine sulfoxide (MetO), gener- ate methyl radicals and modify dC to form 5-methyl-dC in DNA in vitro . We confirmed that both DNA methyla- tion and cancer incidence in the liver were increased by the administration of MetO to oxidatively stressed mice.
In this review, we summarize previous reports on methyl radical generation in vitro and in vivo and DNA modifica- tions by methyl radicals, including our discoveries, as well as our recent experimental evidence suggesting that free radical-mediated dC methylation triggers epigenetic changes.
Keywords: 5-methyldeoxycytidine; DNA methylation;
epigenetic; methyl radical.
*Corresponding author: Hiroshi Kasai , Institute of Industrial Ecological Sciences, Department of Environmental Oncology, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan, e-mail: h-kasai@med.uoeh-u.ac.jp
Kazuaki Kawai and Yun-Shan Li: Institute of Industrial Ecological Sciences , Department of Environmental Oncology, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 , Japan
Introduction
DNA methylation at cytosine C-5 in the 5 ′ -CpG-3 ′ sequence facilitates the organization of mammalian genomes by controlling gene expression in developing embryos (1) . CpG-rich regions, termed CpG islands, are present in the 5 ′ regions of many housekeeping genes and are normally unmethylated (2) . During carcinogenesis,
hypermethylation of the normally unmethylated tumor suppressor gene promoters frequently occurs, which can lead to gene silencing (3 – 5) . In both de novo meth- ylation and maintenance methylation, a methyl group is introduced enzymatically to the C-5 position of cyto- sine by DNA methyltransferases (DNMTs). One of the widely accepted mechanisms of enhanced de novo methylation during carcinogenesis is the overexpres- sion of DNMT3b. For example, the inactivation of tumor suppressor genes during cadmium-induced cell trans- formation is reportedly due to the induction of DNMT3b before the hypermethylation of these genes (6) . DNMT3b polymorphisms are also reportedly correlated with prostate and lung cancer risks (7, 8) . The DNMT geno- type with higher activity is related to a more frequent cancer incidence. For example, a C/T polymorphism in the DNMT3b promoter region results in increased activity and has been identified as a risk factor for lung cancer (9) . However, increased expression of DNMTs is not always correlated with DNA hypermethylation (10) . The detailed molecular mechanism underlying the initial methylation in a normal cell before its carcino- genic transformation is still mostly unknown. Inflam- mation is reportedly involved in DNA hypermethylation and epigenetic changes during carcinogenesis (11) . One of the published hypotheses on the mechanism of de novo DNA methylation, especially in relation to inflam- mation, is the HOCl-induced chlorination of DNA at the cytosine C-5 position or the incorporation of the nucleo- tide product 5-chloro-dCTP into DNA (12) . 5-Chloro-dC in DNA mimics 5-methyldeoxycytidine (m 5 dC) and is recognized by the maintenance DNMT protein, which triggers further DNA methylation. The same considera- tion is also possible for another type of inflammation- induced DNA damage, 5-bromo-dC.
We proposed that during carcinogenesis, the nor- mally unmethylated cytosine C-5 in tumor suppressor gene promoters is initially methylated by a free radical
mechanism, for the following reasons: (1) methyl radi- cals are produced from various environmental agents and endogenous compounds, including methionine sul- foxide (MetO) in the presence of Fe 2 + or oxygen radicals in vitro (for further information, see the next section).
(2) Methyl radicals are produced by various well-known carcinogens or tumor promoters, such as dimethylhydra- zine (DMH), acetaldehyde, tertiary -butyl hydroperoxide (tBuOOH), and cumene hydroperoxide (CuOOH), by their metabolism. (3) DNA methylation inducers, including inflammation (11) , aging (13) , and smoking (14) , as well as many epi-mutagens, such as nickel, chromate, arsenite, hydroxy urea, cisplatin, and diethylstilbestrol, directly or indirectly generate oxygen radicals (15 – 17) and may trigger the formation of methyl radicals from the endogenous molecule, MetO. (4) The cytosine C-5 position is targeted by free radical reactions, based on quantum chemical calculations (18) .
Based on these previous findings, it was tempting to test the reaction of dC with the methyl radical-producing tumor promoters tBuOOH and CuOOH in the presence of Fe 2 + because they are known to methylate deoxyguano- sine (dG) at the C-8 position by a free radical mechanism.
The formation of m 5 dC was clearly demonstrated in these in vitro reactions, although the yield was lower than that of methylated dG, 8-methyldeoxyguanosine (m 8 dG) (19) (Figure 1). Oxidized methionine, MetO, also generated methyl radicals in the presence of hydroxyl radical ( · OH) and induced the dC → m 5 dC modification (20) . Increased MetO formation in proteins has been detected in conjunc- tion with inflammation, aging, and smoking (21 – 24) , thus suggesting a link between free radical-mediated dC meth- ylation and epigenetic changes during carcinogenesis (25) .
In this review, we summarize the previous reports on the generation of methyl radicals in vitro and in vivo , the DNA modifications induced by methyl radicals, including our discoveries, and our recent experimental evidence suggesting that free radical-mediated dC methylation trig- gers epigenetic changes.
Figure 1 Formation of m 5 dC via a methyl radical by DMSO and MetO and by tBuOOH and CuOOH.
Generation of methyl radicals and their reactions with nucleic acid components
Various examples of the generation of methyl radi- cals from endogenous and exogenous compounds and their reactions with nucleic acid components have been reported. In many cases, reactive oxygen species (ROS) have been implicated as essential factors in methyl radical production from chemicals, and iron is often involved in ROS production.
tBuOOH produces methyl radicals by metal catalysis.
Maeda et al. (26) first reported the methyl radical-medi- ated modification of nucleic acid components. Guanine, guanosine, and 5 ′ -guanilic acid were methylated with tBuOOH in the presence of ferrous ion to yield the cor- responding 8-methyl derivatives, especially under acidic conditions. Adenine and adenosine also underwent meth- ylation to produce the C2- or C8-methyl derivatives. Hix et al. (27) also reported methyl radical generation from the tBuOOH-Fe 2 + system and adduct formation, includ- ing m 8 dG, in DNA. The reaction of a hydroxyl radical with t -butyl alcohol, the major metabolite of the carcino- genic gasoline additive methyl t -butyl ether, produces the alkoxyl radical, which can undergo spontaneous fission to produce acetone and the methyl radical (28) . The human carcinogen acetaldehyde generates a methyl radical upon treatment with peroxynitrite and Fe 2 + /H 2 O 2 (29) . Further- more, Kang et al. (30) reported that methyl radicals were generated from dimethylsulfoxide (DMSO), with hydroxyl radicals produced from a Fenton-type reaction between hydrogen peroxide and iron(II)-EDTA. When RNA was reacted with this system, 8-methylguanine, 2-methylad- enine, and 8-methyladenine were detected in the acid hydrolysate. Makino (31) reported methyl radical produc- tion in a γ -irradiated aqueous solution of dl -methionine, as detected by ESR spectroscopy. Nakao et al. (32) reported that both free and peptide-bound MetO are oxidized by hydrogen peroxide/iron(II)-EDTA and peroxynitrite, through a hydroxyl radical intermediate, to produce a methyl radical.
Various descriptions of methyl radical formation in vitro in relation to AGE (advanced glycation end- product), a methylcobalt compound, and a biochemical cofactor have been published. Nakayama et al. (33) reported that the free radicals generated by methylglyoxal (an AGE compound) and hydrogen peroxide included the methyl radical. Kofod (34) reported that pentaaminemethylcobalt (III) decomposes upon dissolution in water by releasing a
methyl radical, which reacts with AMP and GMP to afford 8-methyl-AMP and 8-methyl-GMP, respectively. Methyl radical generation was suggested during methyl transfer from methylcobalamin, a cofactor involved in biomethyla- tion reactions such as the methylations of homocysteine and heavy metals, to diaquocobinamide (35) .
Methyl radicals are also generated by the metabolism of chemicals. In the horseradish peroxidase-catalyzed oxi- dation of the colon carcinogen 1,2-DMH, the ESR spectra of the 2-methyl-2-nitrosopropane spin-trapped radicals revealed the methyl radical (36) . Additionally, the metabo- lism of DMH by liver microsomes revealed methyl radical generation in vitro (37) .
Several examples of methyl radical formation in vivo or in cultured cells have been published. Nakao et al. (38) performed EPR spin trapping studies and reported that acetaldehyde metabolism by xanthine oxidase, submito- chondrial particles, and in whole rats generates methyl radicals. An α -(4-pyridyl-1-oxide)- N - tert -butylnitrone (POBN)-methyl radical adduct was also detected in the bile of acetaldehyde-treated rats. 8-Methylguanine was found in the liver and colon DNA of rats treated with DMH (39) . The anticancer hydrazine derivative procarbazine, which also has carcinogenic properties, generates methyl radicals in rat organs (40) . When tBuOOH was adminis- tered to rats, methyl radical generation was detected by the EPR spin trapping technique, and methyl radical-dG adduct formation was observed in the liver and stomach DNA (41) . Methyl radicals were also detected when iso- lated mouse keratinocytes were treated with the skin tumor promoters, tBuOOH and CuOOH, in the presence of spin traps (42) . Evidence for the metabolism of these organic peroxides into methyl radicals by human car- cinoma skin keratinocytes was obtained by an ESR spin trapping study (43) .
To determine whether a skin tumor promoter could induce radical methylation of DNA in vivo , mouse skin was treated with CuOOH in the presence of Fe 2 + , and the meth- ylated purine nucleosides in the epidermal DNA were ana- lyzed by an LC-MS-MS method (44) . 8-Methyldeoxyadeno- sine (m 8 dA) and m 8 dG were detected at levels of 2.65 ± 1.41 and 0.98 ± 0.14 per 10 7 nucleotides, respectively, suggesting that radical DNA methylations, including the formation of m 5 dC, may occur in cellular DNA. These results are com- patible with our hypothesis that de novo DNA methylation at cytosine C-5, triggered by a free radical mechanism, may be related to epigenetic changes in carcinogenesis.
Regarding nucleic acid modifications by methyl radi- cals, only the formation of 8-methylguanine, 2-methyl- adenine, and 8-methyladenine has been reported. The generation of 5-methylcytosine has not been detected,
possibly due to its low yield. In vivo , a small increase of 5-methylcytosine in DNA would be especially difficult to detect because it is a normal DNA constituent.
Methylation at the C-5 position of cytosine by a methyl radical
The C-5 position of cytosine can be methylated by envi- ronmental chemicals because it is an active site for free radical reactions (18) . We found m 5 dC as a reaction product of CuOOH and deoxycytidine (dC) under physiological conditions (pH 7.4) in the presence of ferrous ion (19) (Figure 1). m 5dC was also formed by the reaction of tBuOOH and dC under the same conditions. As CuOOH and tBuOOH are both tumor promoters, these non-enzy- matic methylation reactions are particularly interesting.
These chemicals are widely used in industry. CuOOH is a synthetic intermediate for acetone and phenol produc- tion, and tBuOOH is an initiator for radical polymeriza- tion. Therefore, humans are exposed to these organic per- oxides during manufacturing processes (45) . It is worth mentioning that m 5 dC formation by CuOOH was strongly inhibited by the free radical scavengers 2,2,6,6-tetrameth- ylpiperidine-1-oxyl and POBN. This finding supports the idea that cytosine methylation occurs via methyl radical generation from environmental chemicals, such as CuOOH or tBuOOH. The rate of dC methylation in DNA by CuOOH is about 10 times slower than that of monomeric dC under the same conditions [63 m m CuOOH and 6 m m Fe(II) at pH 7.4] (19) . This rate difference is probably due to the steric hindrance of double-stranded DNA, which often occurs in chemical modifications of DNA. For example, when dG, as the monomer or in DNA, was reacted with oxygen radicals, a 16-fold higher yield of 8-hydroxydeoxy- guanosine (8-OHdG) from dG was observed, as compared with its formation in DNA (46) .
The formation of m 5 dC from dC or in DNA in vitro was also observed in treatments with DMSO and MetO, under physiological conditions at pH 7.3 in the presence of the Fenton reagent (20) . The production levels of m 5 dC in the DNA-100 m m DMSO-Fenton and DNA-100 m m MetO-Fen- ton reactions were 5/10 4 dC and 1.6/10 4 dC, respectively. In addition, m 8 dG was detected in DNA under the same reac- tion conditions.
We recently confirmed the increases in DNA methyla- tion and cancer incidence by the administration of MetO to mice (47) . In that study, instead of the Fenton reaction in vitro , non-alcoholic steatohepatitis (NASH) mice were used for higher ROS production. NASH mice develop hepatitis
caused by endogenous oxidative stress. The incidences of hepatocellular carcinoma were 16.7 % , 80 % , and 90 % in the groups provided drinking water containing 0 % , 2 % , and 3 % MetO, respectively (Figure 2 ). The DNA methylation status of the p16 gene promoter region was higher in the liver of the MetO-treated mice (in both the non-tumor region and the liver tumor) (Figure 3 ). The higher incidence of hepatocarci- noma may be due in part to the m 5 dC formation by methyl radicals in vivo . In addition, we confirmed the formation of m 8 dA in the MetO-treated mouse liver DNA, suggesting that methyl radicals are actually produced in the mouse liver by those treatments (44) . Notably, MetO is generated in pro- teins by inflammation, aging, and smoking (21 – 24) , which are recognized as DNA hypermethylation inducers (11, 13, 14) . In addition, MetO reductase (Msr), which repairs MetO in proteins, is reportedly a tumor suppressor (48) .
Discussion
The hypermethylation of the tumor suppressor gene pro- moter DNA is considered to play an important role in car- cinogenesis. Clark and her collaborators (49) suggested that the hypermethylation of the GSPT1 promoter is initi- ated by a combination of transcriptional gene silencing and methylation seeds. However, the precise mechanism of the initial seeding methylation is not known. It was also reported that de novo (initial) methylation occurs in
0%
1/6
8/10
9/10
2% 3%
0 20 40 60 80 100
Incidence (%)
MetO
Figure 2 Incidence of hepatocellular carcinoma after administra- tion of MetO to NASH mice.
The numbers above the bar represent the numbers of tumor bearing mice/total number of mice.
U M U M U M
MetO 0% 3% 3%
Noncancerous region Hepatocellular carcinoma
Figure 3 Methylation of p16 in liver tumor and non-tumor regions of NASH mice after treatment with MetO.
nucleosome-free linker regions, which can be accessed by DNMTs (50) . Ushijima and his collaborators (51) reported that RNA polymerase II (pol II) binding around a transcrip- tion start site can function as a protective factor for DNA methylation. They speculated that pol II forms a complex with transcription factors, and its binding around promoter CpG islands may compete with DNMTs. If we consider the free radical mechanism for seeding methylation, instead of enzymatic methylation, then site-specific methylation may also depend on the open or closed structure of DNA, due to protein binding. For example, we observed that 8-OHdG, a form of oxidative DNA damage induced by an OH radical, was not formed and distributed uniformly throughout the whole genome but was restricted to particular genes and regions, depending upon the chromatin structure (52) .
Oxidative stress seems to play an important role in epi- genetic changes during carcinogenesis (53 – 55) . Consider- ing the mitochondrial localization of high concentrations of oxygen radicals and MetO (56) , the mitochondria (mt) might be a potential source of methyl radical generation, involved in cytosine C-5 methylation. In fact, enhanced cytosine methylation of liver mtDNA in selected regions of the mt genome, such as the mitochondrially encoded NADH dehydrogenase 6 gene, and its decreased expres- sion have been implicated in the pathogenesis of NASH (57) . DNMT1 is also reportedly present in the mitochon- drial matrix, where it methylates mtDNA, suggesting the presence of epigenetic modifications of the mitochondrial genome in the regulation of mitochondrial transcription (58) . In addition, insulin resistance in non-alcoholic fatty liver disease is epigenetically regulated by the methyla- tion of the peroxisome proliferation-activated receptor γ coactivator 1 α promoter and is accompanied by mitochon- drial dysfunction (59) .
Bernal et al. (60) recently reported that a low-dose ionizing radiation (0.7 – 7.6 cGy) exposure during gestation significantly increased DNA methylation at viable yellow agouti (A vy ) locus in male offspring. Their coat color, which is epigenetically controlled, was concomitantly shifted from yellow to pseudoagouti. Maternal dietary antioxi- dant supplementation mitigated both the DNA methyla- tion changes and coat color shift. These results suggested that ionizing radiation elicits epigenetic changes medi- ated by oxidative stress. It is reasonable to speculate that the initial seeds of methylation are formed by a free radical mechanism because methyl radicals are generated by the γ -irradiation of methionine, an endogenous molecule (31) . Our hypothesis for free radical-mediated m 5 dC forma- tion, in conjunction with a model of DNA hypermethylation during carcinogenesis by Stirzaker et al. (49) , is shown in Scheme 1 . Even if the level of free radical-mediated m 5 dC
formation is very low, it will gradually increase because a specific repair system for m 5 dC has not been found. Methyl radicals are presumably able to modify cytosine in any of the CpG, CpA, CpC, and CpT sequences. However, only methylated CpG is recognized by DNMT1, and the meth- ylation patterns can be maintained from the parental to the newly synthesized daughter DNA strands in DNA repli- cation, whereas m 5 dC in other sequences would disappear after DNA replication. In addition, only methylated CpG in a specific sequence context can be recognized by methyl- CpG binding domain proteins (MBD2 and MeCP2), which
recruit DNMT3 and histone deacetylase (61) . These pro- teins promote de novo methylation spreading and deacet- ylation, respectively. Finally, dense DNA methylation and stronger gene silencing may occur. Therefore, an increase in non-specific cytosine C-5 methylation seeds may enhance the probability of functionally important meth- ylation changes. Natural selection, instead of molecular targeting, is also involved in the formation of gene- and tissue-specific methylation patterns in cancer cells (62) . Certain CpG islands are hypermethylated in cancer cells, whereas others are not because the hypermethylation confers a selective advantage to the survival or growth of the cancer cells.
In addition to the DNA hypermethylation of tumor suppressor gene promoters, global hypomethylation is also often observed in cancer cell DNA. Under oxidative stress conditions, oxygen radicals result in the oxidation of dG to 8-OHdG and m 5 dC to 5-hydroxymethyldeoxycy- tidine (5-hmdC) in DNA. The formation of either 8-OHdG or 5-hmdC in the m 5 CpG sequence inhibits the DNMT1- mediated methylation of cytosine in the opposite DNA strand, thus generating global hypomethylation (12, 63) . Therefore, both the promoter hypermethylation and the global hypomethylation can be explained by a free radical mechanism. Our studies suggested that free radical-medi- ated cytosine C-5 methylation is a possible mechanism of seeding methylation. Our experimental data revealed that oxygen radicals exert epigenetic as well as muta- genic changes. It should be emphasized that there are no contradictions between our hypothesis and the presently accepted concepts of cancer epigenetics.
Received November 22, 2012; accepted January 18, 2013
References
1. Razin A, Cedar H. DNA methylation and gene expression.
Microbiol Rev 1991; 55: 451 – 8.
2. Cerda S, Weitzman SA. Influence of oxygen radical injury on DNA methylation. Mutat Res 1997; 386: 141 – 52.
3. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer 2004; 4: 43 – 153.
4. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349:
2042 – 54.
5. Lewandowska J, Bartoszek A. DNA methylation in cancer development, diagnosis and therapy-multiple opportunities for genotoxic agents to act as methylome disruptors or remediators, Mutagenesis 2011; 26: 475 – 87.
6. Benbrahim-Tallaa L, Waterland RA, Dill AL, Webber MM, Waalkes MP. Tumor suppressor gene inactivation during cadmium-induced malignant transformation of human prostate cells correlates
with overexpression of de novo DNA methyltransferase. Environ Health Perspect 2007; 115: 1454 – 9.
7. Singal R, Das PM, Manoharan M, Reis IM, Schlesselman JJ. Polymorphisms in the DNA methyltransferase 3b gene and prostate cancer risk. Oncol Rep 2005;
14: 569 – 73.
8. Lee SJ, Jeon HS, Jang JS, Park SH, Lee GY, Lee BH, Kim CH, Kang YM, Lee WK, Kam S, Park RW, Kim IS, Cho YL, Jung TH, Park JY. DNMT3B polymorphisms and risk of primary lung cancer. Carcinogenesis 2005; 26: 403 – 9.
9. Shen H, Wang L, Spitz MR, Hong WK, Mao L, Wei Q. A novel polymorphism in human cytosine DNA-methyltransferase-3B promoter is associated with an increased risk of lung cancer.
Cancer Res 2002; 62: 4992 – 5.
10. Fang CY, Sun WQ, Han HR, Shi LH, Wang L, Zhao Y, Tan Y. The -149C > T polymorphism of DNMT3B is not associated with Endogenous &
exogenous compounds Metabolism
Inflammation oxidative stress
Seeding DNA methylation in protein-free regions Gene inactivation
MBD2 binds to m5dC seeds Recruits HDAC
Recruits DNMT Methylation spread to adjacent regions Natural selection for growth advantage
Sequence specific & gene specific DNA hypermethylation
Normal cell
Cancer cell
• CH3
• OH
•
•
Scheme 1 Free radical-mediated m 5 dC formation and DNA hyper- methylation during carcinogenesis.
colorectal cancer risk: evidence from a meta-analysis based on case-control studies. Exp Ther Med 2012; 4: 728 – 32.
11. Suzuki H, Toyota M, Kondo Y, Shinomura Y. Inflammation-related aberrant patterns of DNA methylation: detection and role in epigenetic deregulation of cancer cell transcriptome. Methods Mol Biol 2009; 512: 55 – 69.
12. Valinluck V, Sowers LC. Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyl- transferase DNMT1. Cancer Res 2007; 67: 946 – 50.
13. Kim SK, Jang HR, Kim JH, Kim M, Noh SM, Song KS, Kang GH, Kim HJ, Kim SY, Yoo HS, Kim YS. CpG methylation in exon 1 of transcription factor 4 increases with age in normal gastric mucosa and is associated with gene silencing in intestinal-type gastric cancers. Carcinogenesis 2008; 29: 1623 – 31.
14. Kim JS, Kim H, Shim YM, Han J, Park J, Kim DH. Aberrant methylation of the FHIT gene in chronic smokers with early stage squamous cell carcinoma of the lung. Carcinogenesis 2004; 25: 2165 – 71.
15. Henkler F, Brinkmann J, Luch A. The role of oxidative stress in carcinogenesis induced by metals and xenobiotics. Cancer 2010; 2: 376 – 96.
16. Klein CB, Costa M. DNA methylation, heterochromatin and epigenetic carcinogens. Mutat Res 1997; 386: 163 – 80.
17. Nyce JW. Drug-induced DNA hypermethylation: a potential mediator of acquired drug resistance during cancer chemotherapy. Mutat Res 1997; 386: 153 – 61.
18. Kochetkov NK, Budovskii EI, Sverdlov ED, Simukova NA, Turchinskii MF, Shibaev VN. Organic chemistry of nucleic acid:
part A. London: Plenum Press, 1971: 166 – 71
19. Kasai H, Kawai K. DNA methylation at the C-5 position of cytosine by methyl radicals: a possible role for epigenetic change during carcinogenesis by environmental agents. Chem Res Toxicol 2009; 22: 984 – 9.
20. Kawai K, Li YS, Song MF, Kasai H. DNA methylation by dimethyl sulfoxide and methionine sulfoxide triggered by hydroxyl radical and implications for epigenetic modifications. Bioorg Med Chem Lett 2010; 20: 260 – 5.
21. Hosako M, Ogino T, Omori M, Okada S. Cell cycle arrest by monochloramine through the oxidation of retinoblastoma protein. Free Radic Biol Med 2004; 36: 112 – 22.
22. Onorato JM, Thorpe SR, Baynes JW. Immunohistochemical and ELISA assay for biomarkers of oxidative stress in aging and disease. Ann NY Acad Sci 1998; 854: 277 – 90.
23. Wells-Knecht MC, Lyons TJ, McCance DR, Thorpe SR, Baynes JW. Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes.
J Clin Invest 1997; 100: 839 – 46.
24. Carp H, Miller F, Hoidal JR, Janoff A. Potential mechanism of emphysema: α 1 -proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. Proc Natl Acad Sci USA 1982; 79: 2041 – 5.
25. Kasai H, Kawai K, Li YS. DNA methylation at the C-5 position of cytosine by a methyl radical: a link between environmental agents and epigenetic change. Genes Environ 2011; 33: 61 – 5.
26. Maeda M, Nushi K, Kawazoe Y. Studies on chemical alterations of nucleic acids and their components. VII. C alkylation of purine bases through free radical process catalyzed by ferrous ion.
Tetrahedron 1974; 30: 2677 – 82.
27. Hix S, Morais MD, Augusto O. DNA methylation by tert-butyl hydroperoxide-iron (II). Free Radic Biol Med 1995; 19: 293 – 301.
28. McGregor DB, Cruzan G, Callander RD, May K, Banton M. The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetateTM and methyl tertiary-butyl ether in Salmonella typhimurium. Mutat Res 2005; 565: 181 – 9.
29. Nakao LS, Ouchi D, Augusto O. Oxidation of acetaldehyde by peroxynitrite and hydrogen peroxide/iron(II). Production of acetate, formate, and methyl radicals. Chem Res Toxicol 1999;
12: 1010 – 8.
30. Kang JO, Gallagher KS, Cohen G. Methylation of RNA purine bases by methyl radicals. Arch Biochem Biophys 1993; 306:
178 – 82.
31. Makino K. Studies on spin-trapped radicals in γ -irradiated aqueous solutions of DL-methionine by high performance liquid chromatography and ESR spectroscopy. J Phys Chem 1979; 83:
2520 – 3.
32. Nakao LS, Iwai LK, Kalil J, Augusto O. Radical production from free and peptide-bound methionine sulfoxide oxidation by peroxynitrite and hydrogen peroxide/iron (II). FEBS Lett 2003;
547: 87 – 91.
33. Nakayama M, Saito K, Saito E, Nakayama K, Terawaki H, Ito S, Kohno M. Radical generation by non-enzymatic reaction of methylglyoxal and hydrogen peroxide. Redox Rep 2007; 12:
126 – 33.
34. Kofod P. GMP and AMP as methyl radical traps in the reaction with pentaamminemethylcobalt (III). J Inorg Biochem 2004; 98:
1978 – 80.
35. Fanchiang YT, Bratt GT, Hogenkamp HPC. Methyl transfer from methylcobalamin to diaquocobinamide. Proc Natl Acad Sci USA 1984; 81: 2698 – 702.
36. Augusto O, Du Plessis LR, Weingrill CLV. Spin-trapping of methyl radical in the oxidative metabolism of 1,2-dimethylhydrazine.
Biochem Biophys Res Commun 1985; 110: 625 – 31.
37. Albano E, Tomasi A, Goria-Gatti L, Iannone A. Free radical activation of monomethyl and dimethyl hydrazines in isolated hypatocytes and liver microsomes. Free Radic Biol Med 1989; 6:
3 – 8.
38. Nakao LS, Kadiiska MB, Mason RP, Grijalba MT, Augusto O.
Metabolism of acetaldehyde to methyl and acetyl radicals: in vitro and in vivo electron paramagnetic resonance spin-trapping studies. Free Radic Biol Med 2000; 29: 721 – 9.
39. Netto LES, RamaKrishna NVS, Kolar C, Cavalier EL, Rogan EG, Lawson TA, Augusto O. Identification of C8-methylguanine in the hydrolysates of DNA from rats administered 1,2-dimethylhy- drazine. Evidence for in vivo DNA alkylation by methyl radicals. J Biol Chem 1992; 267: 21524 – 7.
40. Goria-Gatti L, Iannone A, Tomasi A, Poli G, Albano E. In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin-trapping study. Carcinogenesis 1992; 13:
799 – 805.
41. Hix S, Kadiiska MB, Mason RP, Augusto O. In vivo metabolism of tert-butyl hydroperoxide to methyl radicals. EPR spin-trapping and DNA methylation studies. Chem Res Toxicol 2000; 13:
1056 – 64.
42. Taffe BG, Takahashi N, Kensler TW, Mason RP. Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes, formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydrop- eroxide. J Biol Chem 1987; 262: 12143 – 9.
43. Athar M, Mukhtar H, Bickers DR, Khan IU, Kalyanaraman B. Evidence for the metabolism of tumor promoter organic hydroperoxides into free radicals by human carcinoma skin keratinocytes: an ESR-spin trapping study. Carcinogenesis 1989; 10: 1499 – 503.
44. Kasai H, Kawai K, Song MF, Li YS, Hattori T, Matsuda T. Analyses of 8-methyldeoxyadenosine and 8-methyldeoxyguanosine as markers of free radical-mediated DNA methylation in mouse.
Genes Environ 2013; 35: 5–9.
45. Murray AR, Kisin ER, Kommineni C, Vallyathan V, Castranova V, Shvedova AA. Pro/antioxidant status and AP-1 transcription factor in mouse skin following topical exposure to cumene hydroperoxide. Carcinogenesis 2007; 28: 1582 – 8.
46. Kasai H, Nishimura S. Hydroxylation of deoxyguanosine at C-8 position by ascorbic acid and other reducing agents. Nucl Acids Res 1984; 12: 2137 – 45.
47. Kawai K, Li YS, Song MF, Ootsuyama Y, Kakehashi A, Wanibuchi H, Ootsuyama A, Norimura T, Kasai H. Methionine sulfoxide stimulates hepatocarcinogenesis in non-alcoholic steato- hepatitis (NASH) mouse: possible role of free radical-mediated DNA methylation. Genes Environ 2012; 34: 123 – 8.
48. Lei KF, Wang YF, Zhu XQ, Lu PC, Sun BS, Jia HL, Ren N, Ye QH, Sun HC, Wang L, Tang ZY, Qin LX. Identification of MSRA gene on chromosome 8p as a candidate metastasis suppressor for human hepatitis B virus-positive hepatocellular carcinoma. BMC Cancer 2007; 7: 172.
49. Stirzaker C, Song JZ, Davidson B, Clark SJ. Transcriptional gene silencing promotes DNA hypermethylation through a sequential change in chromatin modifications in cancer cells. Cancer Res 2004; 64: 3871 – 7.
50. Hinshelwood RA, Melki JR, Huschtscha LI, Pail C, Song JZ, Stirzaker C, Reddel RR, Clark SJ. Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodeling and mimics nucleosome positioning. Hum Mol Genet 2009; 18: 3098 – 109.
51. Takeshima H, Yamashita S, Shimizu T, Niwa T, Ushijima T. The presence of RNA polymerase II, active or stalled, predicts epigenetic fate of promoter CpG islands. Genome Res 2009; 19:
1974 – 82.
52. Nomoto M, Yamaguchi R, Kawamura M, Kohno K, Kasai H.
Analysis of 8-hydroxyguanine in rat kidney genomic DNA after
administration of renal carcinogen, ferric nitriloacetate. Carcino- genesis 1999; 20: 837 – 41.
53. Ziech D, Franco R, Pappa A, Panayiotidis MI. Reactive oxygen species (ROS)-induced genetic and epigenetic alterations in human carcinogenesis. Mutat Res 2011; 711: 167 – 73.
54. Cyr AR, Domann FE. The redox basis of epigenetic modifications:
from mechanisms to functional consequences. Antioxid Redox Signal 2011; 15: 551 – 89.
55. Donkena KV, Young CYF, Tindall DJ. Oxidative stress and DNA methylation in prostate cancer. Obstet Gynecol Int 2010; 2010:
article ID 302051.
56. Ugarte N, Petropoulos I, Friguet B. Oxidized mitochondrial protein degradation and repair in aging and oxidative stress.
Antioxid Redox Signal 2010; 13: 539 – 49.
57. Pirola CJ, Fern á ndez Gianotti T, Burgue ñ o AL, Rey-Funes M, Loidl CF, Mallardi P, San Martino J, Casta ñ o GO, Sookoian S.
Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease. Gut 2012; PMID 22879518 [Epub ahead of print].
58. Shock LS, Thakkar PV, Peterson EJ, Moran RG, Taylor SM.
DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria. Proc Natl Acad Sci USA 2011; 108: 3630 – 5.
59. Sookoian S, Rosselli MS, Gemma C, Burgueno AL, Gianotti TF, Castano GO, Pirola CJ. Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1 α promoter. Hepatology 2010; 52: 1992 – 2000.
60. Bernal AJ, Dolinoy DC, Huang D, Skaar DA, Weinhouse C, Jirtle RL.
Adaptive radiation-induced epigenetic alterations mitigated by antioxidants. FASEB J 2012; PMID 23118028 [Epub ahead of print].
61. Clouaire T, de Las Heras JI, Merusi C, Stancheva I. Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA. Nucl Acids Res 2010; 38:
4620 – 34.
62. Esteller M. Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum Mol Genet 2007; 16: R50 – 9.
63. Turk PW, Laayoun A, Smith SS, Weitzman SA. DNA adduct 8-hydroxy-2 ′ -deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase. Carcinogenesis 1995; 16: 1253 – 5.
Yun-Shan Li received her PhD from the University of Okayama, Japan, in 2006. She is a research associate at the Department of Environmental Oncology, University of Occupational and Environmental Health, Japan, since 2006.
Kazuaki Kawai received his PhD from the University of Shizuoka, Japan, in 1998. He became an associate professor and profes- sor at the Department of Environmental Oncology, University of Occupational and Environmental Health, Japan, in 2003 and 2012, respectively.
Hiroshi Kasai was the section chief of the Biology Division, National Cancer Center Research Institute, Tokyo, Japan, from 1988 to 1992.
He then became a professor at the Department of Environmental Oncology, University of Occupational and Environmental Health, Japan, from 1993 to 2012.
